Dr. Kirby C. Stafford III Department of Entomology The Connecticut Agricultural Experiment Station New Haven, CT 06504 Evaluation of Natural Products for the Control of the Tick Vectors of Lyme Disease Spirochetes Project Abstract The objective of this proposed project is to identify and devise formulations of natural products, particularly the eremophilane sesquiterpene nootkatone, and evaluate their repellency to and effectiveness for the reduction of questing nymphal Ixodes scapularis in the field. We propose several candidate natural low-toxicity chemicals potentially toxic or repellent against I. scapularis. Working with a scientist with the USDA Agricultural Research Service, we will improve the current emulsifiable concentrate (EC) formulation of nootkatone and develop preliminary encapsulation techniques for this chemical using coacervation, Fantesk encapsulation, and spray dried encapsulation. Formulation samples will be screened in the laboratory at The Connecticut Agricultural Experiment Station for efficacy against I. scapularis and the formulations will be optimized for aqueous spray applications. Field trials will be conducted in communities in Fairfield and Litchfield Counties of Connecticut;areas with high tick densities and endemic for Lyme disease. The repellency of the new natural product candidates will be evaluated initially in the laboratory against I. scapularis. In the field, repellency of nootkatone, and other candidate natural products will be evaluated using treated and untreated flannel tick drags. In addition, we will bring several added components to the proposed project beyond the stated objectives in the funding opportunity announcement that will enhance the outputs and outcomes of the research. Chemical analysis of field samples will provide nootkatone residue-degradation data. In addition, we propose to further test the compatibility and field efficacy of the entomopathogenic fungus Metarhizium anisopliae Strain 52 with nootkatone and other natural products, which may permit effective use of lower concentrations of the natural compounds in the field for a more economical and integrated natural approach to tick control. PI: Dr. Kirby C. Stafford, III Vice Director, Chief Entomologist, State Entomologist Department of Entomology The Connecticut Agricultural Experiment Station 123 Huntington Street, P.O. Box 1106, New Haven, CT 06504 Tel. (203) 974-8485 Fax (203) 974-8502 E-mail: Kirby.Stafford@po.state.ct.us PROJECT NARRATIVE Evaluation of Natural Products for the Control of the Tick Vectors of Lyme Disease Spirochetes A. SPECIFIC AIMS The aims of this proposed project are to identify and devise formulations of natural products, particularly the eremophilane sesquiterpene nootkatone, and evaluate their effectiveness for the reduction of questing nymphal Ixodes scapularis in the field in areas highly endemic for Lyme disease. This project will assist in developing new technology for the management of tick vectors of Lyme disease spirochetes, determine how to integrate natural botanical compounds with a biological control agent, and ultimately provide an alternative tick management option to conventional acaricides for individual stakeholders and the land care industry. Within the project goal to evaluate the efficacy of natural products for the control of the tick vectors of Lyme disease spirochetes, there are five research objectives. We propose a study on the control of blacklegged ticks using natural products as outlined in the funding opportunity announcement, including but not limited to nootkatone, repellency of these natural products to I. scapularis, and, in addition, test the compatibility and field efficacy of the entomopathogenic fungus Metarhizium anisopliae Strain 52 with nootkatone and other natural products for a more integrated natural and biological approach. People prefer the use of natural compounds or biological agents for pest control and personal protection measures. Availability of natural alternative control materials and agents may lead to increased acceptance of applications of acaricidal agents to the landscape and increased tick control. The objectives are: Objective 1. Identify all-natural, low-toxicity chemicals extracted from botanical sources, including the eremophilane sesquiterpene, nootkatone, toxic to the nymphal stage of the blacklegged tick, I. scapularis, and devise formulations (water miscible, emulsifiable, and microencapsulated or other slow-release formulations to extend residual activity) for aqueous application to vegetation and leaf litter for the control of host-seeking I. scapularis (FOA research objectives 1 &2). Objective 2. Field test formulations against populations (June peak >0.1/m2) of the blacklegged tick in Lyme disease endemic areas and determine whether control of ticks using test chemicals is accomplished by direct toxicity to ticks in field plots via approach outlined in the proposal announcement (FOA research objectives 3 &4). Objective 3. Test repellency of the natural products against I. scapularis in the laboratory and in the field with treated and untreated flannel tick drags (FOA research objective 5). Objective 4. Test formulations of natural products, particularly nootkatone, in the laboratory and field in combination with the entomopathogenic fungus, M. anisopliae Strain 52, for the control of I. scapularis nymphs. These objectives can be broadly divided into product development and field evaluation of natural products for repellency and control of the tick. The field trials will be conducted in communities in Fairfield and Litchfield Counties of Connecticut;areas highly endemic for Lyme disease. We will bring several added components to the proposed project that will enhance the outputs and outcomes of the research. In cooperation with the Department of Analytical Chemistry at The Connecticut Agricultural Experiment Station (CAES), we will provide quantitative assessment of residual nootkatone after application in the field. Working with a scientist with the USDA Agricultural Research Service, slow release formulation(s) to extend nootkatone efficacy will be developed and tested. Preliminary tests show that germination of M. anisopliae is not affected by 0.1% nootkatone. Therefore, M. anisopliae will also be incorporated into the test of natural product formulations to determine if the agents can be integrated into a control program and permit effective use of lower, economical concentrations of the natural compounds. Nootkatone will be obtained from commercial sources and Novozymes Biologicals, Inc. (Salem, VA) will provide the M. anisopliae. B. BACKGROUND AND SIGNIFICANCE Lyme disease (LD) is the most important tick-borne disease in the United States. A record 23,763 human cases were reported in 2002, closely followed by 23,305 in 2005 (Centers for Disease Control and Prevention 2003;Centers for Disease Control and Prevention 2007), which probably represent only about 10- 20% of diagnosed cases. The disease is caused by the bacterium, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt &Brenner, which is transmitted by the bite of the blacklegged tick, I. scapularis Say. There is a strong correlation between the abundance of infected I. scapularis, specifically the nymphal stage that is most abundant in spring and summer months (Mather et al. 1996;Stafford et al. 1998), and the incidence of LD. Furthermore, this vector is responsible for transmitting the agents of human babesiosis and human granulocytic anaplasmosis, two emerging tick-borne infections in the US. Connecticut has the highest or among the highest rates for LD in the United States, with a rate of 136 cases reported per 100,000 persons in 2002 (4,631 reported cases) and 53 cases per 100,000 population in 2005 (1,810) (Ertel and Nelson 2003;Ertel et al. 2006). The drop in cases from 2003 is largely because of changes in reporting. Fairfield County has the greatest number of cases, generally accounting for about a third of Connecticut's total and Litchfield County has the highest rate of LD (299 per 100,000 population). In addition, there were 49 confirmed and 154 probable cases of anaplasmosis and 69 confirmed cases of human babesiosis in the state in 2002 (Ertel et al. 2003;Ertel et al. 2003). In the northeastern states, individuals with the highest risk for tick bites and Lyme disease are those residing in suburban residential developments with adjacent wooded tracts, and those with rural homes in a woodland environment, where hosts for the tick flourish (Falco and Fish 1988;Falco and Fish 1988; Stafford and Magnarelli 1993). Cases occur most commonly in children aged 5- 14 years and those 45-54 years, which probably reflects outdoor activity in the summer months when nymphal ticks are active. Most cases of LD appear to be acquired peridomestically. Therefore, control of ticks in residential locations, particularly in areas of high disease incidence, are expected to reduce the number of local LD cases. A variety of vector control approaches have been explored to determine their efficacy in reducing tick abundance (Stafford and Kitron 2002;Ginsberg and Stafford III 2005). These include personal protective measures, host management, habitat modification, acaricide applications, host-targeted acaricides, and biological control. While most of these approaches have met with mixed success and acaricides can provide excellent tick control over relatively large areas, environmental concerns have restricted their acceptance and broad use. The acaricidal treatment of important hosts, such as white-footed mice and white-tailed deer also has a substantially reduced environmental impact and has also been shown to reduce tick abundance. Recently completed trials of the rodent bait box in southwestern Connecticut by CAES have shown reduced tick densities at residential home sites though little impact was observed at homes in more rural northwestern parts of the state. However, the commercially developed rodent bait box containing fipronil is no longer being manufactured due to cost issues. The 4-poster technology for the passive application of an acaricide to deer, though providing significant tick control (Pound et al. 2000;Carroll et al. 2002;Carroll and Kramer 2003;Solberg et al. 2003), has not been widely adopted in the northeast for control of the blacklegged tick amid cost issues, placement restrictions, and concerns by wildlife agencies of feeding the corn bait to deer and emerging chronic wasting disease of deer. Acceptance of chemical acaricides in some communities is low. For instance, a study of residents in the towns of Westport and Weston, CT in which Lyme disease attitudes and behaviors were surveyed (under CDC cooperative agreements to the Connecticut Department of Public Health and CAES) found that only 22.5% of residents reported having sprayed a chemical pesticide for tick control, and 69.5% indicated that the use of pesticides was not very likely or likely at all. Several years of community education on how to target and minimize pesticide use has increased use of acaricides to 38% of survey respondents (CT DPH, unpublished data). Yet clearly, there is a strong niche for alternative control methods or compounds and new tools are needed. Biological agents such as entomopathogenic nematodes and parasitoid wasps have had limited success in reducing numbers of I. scapularis. Engorged I. scapularis females are susceptible to fatal infection by steinernematid nematodes, but unfed females and younger (immature) stages are not. Furthermore, sensitivity of nematodes to low fall season temperatures in the northeastern US limits their potential application for control of this tick at present. Likewise, the blacklegged tick is parasitized by the chalcid wasp, Ixodiphagus hookeri, but only at high tick and host deer densities. Thus, it is unlikely that a control approach using this wasp species will be successful (Stafford et al. 2003). By contrast, natural products and entomopathogenic fungi could provide safer, organically acceptable tick control. A combination of these strategies may provide the least toxic options for tick control in the residential landscape. People prefer the use of natural compounds or biological agents for pest control and personal protection measures. Public interest in safer alternatives is reflected in a growing interest in and accreditation of landscape professionals in organic land care and the use and acceptance of natural, organic, and herbal products have been on the rise in recent years (Tanneeru 2006;Cunningham 2007). Repellent products containing natural, botanical ingredients have increasingly become available to the public. Botanical compounds have long been known to have repellent and/or toxic properties against arthropods. Until recently, information on the activity of natural products against ticks has been relatively limited. Tick products currently are restricted to those containing d-limonene for flea and tick control on pets, which has little efficacy against I. scapularis (Panella et al. 1997). Not all botanical extracts or compounds are effective against arthropod vectors or may offer some protection against mosquitoes, but not ticks (e.g. citronella). Many botanicals offer only a short duration of protection (3 to 30 minutes), a problem frequently due to high volatility of the compound requiring frequent replacement. Some African plants have strong tick-repellency (Kaaya 2000). USDA-ARS scientists have shown that two American beautyberry, Callicarpa americana, compounds, callicarpenal and intermedeiol, may be effective repellents against I. scapularis (Pons 2007). The active ingredient of extracts of lemon eucalyptus oil, citriodiol, has been shown in a few studies to provide some repellency and protection against tick bite (Gardulf et al. 2004;Garboui and Jaenson 2006;Jaenson et al. 2006), all against I. ricinus, the tick vector for Lyme disease in Europe. There does not appear to be any peer-reviewed studies evaluating the repellency of lemon-eucalyptus against I. scapularis. The extracts or oils of several other plants have also been found to have repellent properties against I. ricinus (Jaenson et al. 2005). The essential oil from the heartwood of Alaska yellow cedar, Chamaecyparis nootkatensis, was found to be a highly effective toxicant against nymphal I. scapularis (Panella et al. 1997). Various components of the essential oil, especially nootkatone, an eremophilane sesquiterpene, were subsequently shown to have 10x greater activity than the oil itself (Panella et al. 2005). However, a 1-2% rate appears necessary for control in the field (Marc Dolan, personal communication), a rate far above the LC50 of 0.0029% (Panella et al. 2005). The nootkatone derived from grapefruit oil and three other natural compounds were found to be highly repellent, comparable to DEET, in a screening assay against I. scapularis (Diethrich et al. 2006). Clearly, there is a large disparity in efficacy of botanical compounds against ticks, ranging from highly effective to no observable effect as a repellent or toxicant. The recognized safety of some compounds (e.g.; citronella and citronella oil, eugenol, garlic and garlic oil, geraniol, geranium oil, lemongrass oil, rosemary and rosemary oil, thyme and thyme oil) by the EPA (40 CFR 152.25 - minimum risk pesticides) and their exemption from the requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) has lead to their use in formulated products of unknown effectiveness against ticks. While no claim to control disease may be made, a few products do claim to control "deer ticks". Active ingredients approved by the Organic Materials Review Institute (OMRI) include neem products, Bacillus thuringiensis (BT), Beauveria bassiana (fungus), garlic, pyrethrin, rosemary oil, and insecticidal soap. Clarification of the efficacy of some compounds and the development of new ones are badly needed. A number of fungi have been isolated from ticks, primarily from engorged females collected off deer, although some fungal species have been found on larvae, nymphs, and unfed females. These include Paecilomyces farinosus, Lecanicillium (formerly Verticillium) lecanii, Beauveria bassiana, and some other species from these three genera in the Hyphomycetes family. Recovery rates are low, however, generally less than 5%. Lecanicillium lecanii appears to be a common fungal pathogen of I. scapularis and I. ricinus in nature. Entomopathogenic fungi such as Beauveria bassiana and Metarhizium anisopliae have been reported to cause mortality in I. scapularis. Two products, BotaniGard (Mycotech, Butte Montana) and Naturalis T &O (Troy Biosciences, Phoenix, Arizona) provided effective tick control in the laboratory and in field trials (S. Allan and K. Stafford, unpublished data), but are not labeled for tick use. Initial field trials in 2002 with the entomopathogenic fungus (Metarhizium anisopliae Strain 52) provided high levels of control (80-85%, K. Stafford, unpublished data). This fungus was provisionally registered by the Environmental Protection Agency (EPA) for tick biocontrol. Metarhizium is a naturally occurring soil fungus that is considered nonpathogenic to mammals. This fungus posses minimal risk to non-target organisms and does not harm many beneficial insects such as honey bees, green lacewings, lady beetles, parasitic Hymenoptera or earthworms at rates used. Field applications with M. anisopliae Strain 52 (Tick- Ex", Novozymes Biologicals, Inc., Salem, VA) in 2007 resulted in around 90% control for several weeks after a second application and overall seasonal control of 77.8% at the higher rate tested (see preliminary studies/progress report). Full registration is now pending. It was clear however that applications must be made during nymphal tick activity. Nootkatone has excellent knock-down properties and the fungus could compliment the natural compound to provide improved tick control. However, nootkatone appears to lack a long lasting effect in the field. Formulation technology can enhance the activity of many pesticide applications by extending storage stability, improving dispersion in the spray tank, and extending residual activity after application. Nootkatone has two adverse characteristics that may specifically be addressed by proper formulation development, and specifically by encapsulation. First, the nootkatone is insoluble in water (Material Safety Data Sheet, Bedoukian Research, Danbury, CT). Second, applications of nootkatone have demonstrated a short period of residual activity (M. Dolan, personal communication). Specific reasons for contributing to the rapid loss of efficacy are not known, but likely are related to loss by volatility or adsorption into the treated substrate (plants, leaf litter, soil, etc.). These losses may be reduced by encapsulation formulations that provide a barrier between nootkatone and the air and/or treated substrate. There is high probability of extending residual activity of nootkatone by the application of the appropriate formulation technology. Many encapsulation techniques including spray drying, polymer encapsulation and coacervation are being researched, often for use in the food industry (Gouin 2004), and these techniques may be suitable for encapsulation of nootkatone. Likewise, when considering integrated applications with entomopathogenic fungi, encapsulating nootkatone may again provide a barrier that will allow mixing higher concentrations of nootkatone with spores for application without inhibiting spore germination. At this time, nootkatone is expensive. If a lower rate of nootkatone acts in combination or synergistically with the fungus, then costs could be reduced. An encapsulated formulation may benefit applications of nootkatone by extending residual activity or allow flexibility to tank mix with fungal agents without detrimental affects on spore germination. Natural products alone or in combination with a Metarhizium formulation could also potentially be used in wild animal host-targeted applications. A natural, integrated tick management program will require a variety of tools that can be used under different circumstances and with different segments of the human population. We propose to develop, test, and refine natural products, especially nootkatone, as well as examine their compatible use with the fungus M. anisopliae (Tick-Ex) against the nymphal stage of the blacklegged tick, I. scapularis. C. PRELIMINARY STUDIES/PROGRESS REPORT Scientists at the Connecticut Agricultural Experiment Station (CAES) have been involved in studies of tick ecology and tick control technologies for 20 years. Some tick control trials and testing of new technology is ongoing or recently completed and has not yet been published. Other specialists in related fields chemistry, mycology, and wildlife biology, are also on staff and available for collaboration or consultation. The principal investigator has a supervisory pesticide application license and the technician on the project has a pesticide application license. Strategies that have been evaluated for tick control, most of which have involved CAES, include habitat modification, burning vegetation (Mather et al. 1993;Stafford 1998), the use of parasitoid wasps that infect I. scapularis (Stafford et al. 1996;Stafford et al. 2003), chemical acaricides (Stafford 1991; Solberg et al. 1992;Curran et al. 1993;Schulze et al. 2000;Schulze et al. 2001), pesticide treatment of rodents and deer (Pound et al. 2000;Rand et al. 2000; Solberg et al. 2003;Dolan et al. 2004), exclusion of deer from defined areas (Daniels et al. 1993;Stafford 1993;Daniels and Fish 1995), and the reduction of vertebrate host numbers (Wilson et al. 1984;Deblinger et al. 1993;Stafford et al. 2003). The PI and CAES staff has extensive experience in conducting and evaluating tick control trials with natural (e.g., pyrethrin) and synthetic acaricides and with entomopathogenic fungi. The application of entomopathogenic fungi to tick habitat or vertebrate hosts to control ticks is a more environmentally acceptable approach than chemical use, and fungi are one of the more promising tick biological control agents that could provide a natural complement to botanical compounds. Initial field trials of a new commercial strain of M. anisopliae in 2002 provided 80-85% control of nymphal I. scapularis (K. Stafford and S. Allan, unpublished data) and led the Environmental Protection Agency (EPA) to provisionally register this product for the control of ticks (Tick-Ex") and other pests. We have continued these evaluations in the laboratory and again in the field in 2007 as product became available. Novozymes Biologicals, Inc. acquired Earth BioSciences, Inc. on September 30, 2006, including the Metarhizium anisopliae insecticide product line. The acquisition according to the press release fits into Novozymes position as a leader in the research, development, and manufacture of biotechnology products and natural pest technologies. The company has made the additional investments necessary to begin production and permit expanded field trials in 2007. Additional studies were needed to examine field efficacy of the emulsifiable concentrate (EC) formulation and timing of application for full registration and integration into tick management program. Field trials to evaluate efficacy and spore longevity in the field were conducted by The Connecticut Agricultural Experiment Station in summer 2007, May through August in northwestern Connecticut (CT). Trials were conducted at lawn edges at home sites in the three towns of Salisbury, Falls Village, and Cornwall in cooperation with the Torrington Area Health District. Treatments plots were established at the perimeters of the properties in tick habitat ranging from 39-116 m2 at home sites (Table 1). The oil- based formulation (Tick-EXTM EC, Novozymes Biologicals, Inc., Salem, Virgina) with a concentration of 3.9x109 cfu/ml was sprayed by a commercial applicator (NaturaLawn of America, Danbury, CT) as directed by the principal investigators using a high volume hydraulic sprayer at two application rates (2.6 fl oz /1000 ft2 "lower rate" and 10.4 fl oz/1000 ft2 "higher rate") at 150 psi. A total of 20 home sites were sprayed - 11 sites were treated with lower rate (3.2x105 cfu/cm2 ) and 9 sites were treated with five times higher rate (1.3x106 cfu/cm2). Based on the number of nymphs/100m2 recovered from these sites from 2003-2006, there were no significant differences in nymphal abundance between the sites in the treatment blocks. Control sites (n = 21) received no treatment. The fungus was applied two times;once May 8th and 9th and again on June 29th and July 2nd. The effective rates (viable spores applied) were 2.0x105 and 8.2x105 cfu/cm2 for low and high rates, respectively, for the 1st application and 2.2x105 and 9.0x105 cfu/cm2 for the low and high rates,respectively, for the 2nd application. After the first application 39.8 and 9.9 % control was observed from the lower and higher rate treated sites, respectively for the approximately 5 weeks post application. The percent controls obtained after the second application from lower and higher rate treated sites were 53.2 and 73.8%, respectively, for the period ending July 30, 2007 and 36.5 and 77.8% for the 8 weeks ending August 20, 2007. However, percent control for the 3 weeks following the 2nd application was 87.1 and 96.1 for the low and high rates, respectively. There was a significant difference in the treatment groups in the number of nymphs collected for the whole sampling season (DF = 2, F = 5.271, P = 0.005). In pair wise multiple comparison procedures, there was a highly significant difference between the nymphs collected from control and lower rate sites (t = 2.888, P = 0.004) as well as between the control and high rate sites (t = 2.319, P = 0.021). No significant difference was observed between the low and high rate sites. Preliminary tests at CAES show that germination of the fungus is not affected by 0.1% nootkatone, but there is inhibition at 1.0% (Bharadwaj and Stafford, unpublished data). As noted previously, a 1-2% rate of nootkatone appears necessary for control in the field (Marc Dolan, personal communication), a rate far above the LC50 of 0.0029% (Panella et al. 2005). It may be possible to use a lower economical rate of nootkatone when combined with M. anisopliae. In the course of the tick control research over the past 20 years, extensive contacts have been developed with the Connecticut Department of Public Health, many local and regional health districts, people and businesses, such as commercial applicators, and many stakeholders, such as homeowners, many of whom have provided access to their properties for tick surveys and control studies over many years. There is strong interest by the public in the continuation of these studies. The health departments will continue to work with us (see letters of support). The principal investigator has provided information on ticks and tick management to stakeholders through fact sheets, talks, lectures, workshops, and most recently, a Tick Management Handbook (see Outreach below). D. RESEARCH DESIGN AND METHODS Objective 1. Identify all-natural, low-toxicity chemicals extracted from botanical sources, including the eremophilane sesquiterpene, nootkatone, toxic to the nymphal stage of the blacklegged tick, I. scapularis, and devise formulations (water miscible, emulsifiable, and microencapsulated or other slow-release formulations to extend residual activity) for aqueous application to vegetation and leaf litter for the control of host-seeking I. scapularis (FOA research objectives 1 &2). Natural products evaluated against I. scapularis must include the eremophilane sesquiterpene, nootkatone, but need not be limited to this chemical. Other possible natural compounds for repellent and toxicity testing include nepetalactone (from catmint, Nepeta cataria) and curcumin with mustard oil (from turmeric, Curcuma longa, occasionally used as a tick repellent in India). Carvacrol, another component of the Alaska yellow cedar extract, has been shown to have significant biological activity against I. scapularis, but has not been tested in the field. Some companies are using botanical or natural materials from the EPA's 25b minimum risk pesticide list in mosquito and tick control products. Products containing these active ingredients are exempt from the requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). While products cannot bear claims to control disease or arthropods carrying specific diseases, including ticks that carry Lyme disease, they are labeled for ticks. Garlic-based products (e.g. Mosquito Barrier and Tick and Flea Solution) are already on the market and being applied for the control of I. scapularis. However, no data on its efficacy is available. The CDC project scientist will participate in the selection and approval of test natural acaricides. We will devise water miscible, emulsifiable formulations for testing the natural products. The current emulsifiable formulation of nootkatone uses d-limonene and EZ-Mulse (non-ionic surfactant for use with citrus terpenes) (M. Dolan, personal communication), but does not appear to possess sufficient residual activity. Absorption by substrate, volatility, and degradation by environmental factors are common problems with a natural material. Dr. Robert Behle, National Center Agricultural Utilization Research, USDA Agricultural Research Service, Peoria, Illinois, will evaluate the current EC for emulsification characteristics (ASTM standard E 1116) and determine if improvements are necessary to establish a standard EC formulation to compare with subsequent formulation samples. Slow/extended release or encapsulated formulation(s) to extend efficacy will be developed and evaluated in the laboratory for field testing in year 2 and 3 of the project. We propose three techniques for encapsulating nootkatone. These are: coacervation, an established technique in industry that uses dilute solutions of carbohydrate and proteins to form a coating that can be cured by crosslinking the shell material (Yeo et al. 2005), the FaneskTM process, developed in the USDA-ARS laboratory, that uses starch and a steam jet to encapsulate oils (Fanta et al. 1999) and spray dryer encapsulation using a polymer to encapsulate the oil emulsion in aqueous phase, then flash drying to form a dry powder (Gouin 2004). Storage stability of formulations for physical characteristics, mixing characteristics, and loss of active agent at quarterly time intervals for up to one year will also be examined and stored samples will be provided for efficacy evaluation against I. scapularis. Efficacy Testing of Nootkatone Formulations - The extended release formulations will be evaluated in comparison with emulsifiable formulations against I. scapularis nymphs in the laboratory using a modification of the disposable pipette method (Barnard et al. 1981;Maupin and Piesman 1994). Nymphs will be exposed to inner surface of the pipette treated with the natural product and extended formulation(s) for 24-h and observed for mortality. LC50 and LC90 will be calculated using probit analysis. Suitable formulations will be candidates for semi-field and field trials. Nymphs of I. scapularis for the bioassays will be obtained by rearing female ticks collected in the field on New Zealand White rabbits in our tick rearing facility at Lockwood Farm, Hamden, Connecticut, as approved by the Experiment Station's Animal Care and Use Committee (IACUC) (Protocol S01-05). Larvae hatching from the eggs laid by the female will be fed on laboratory mice to obtain the nymphs used in the trials. Objective 2. Field test formulations against populations (June peak >0.1/m2) of the blacklegged tick in Lyme disease endemic areas and determine whether control of ticks using test chemicals is accomplished by direct toxicity to ticks in field plots via approach outlined in the proposal announcement. Based on laboratory results, we propose to evaluate selected natural products in the field for acaricidal activity, repellency (see Objective 3), and residual activity against nymphal I. scapularis, particularly encapsulated formulations of nootkatone. Field Applications - The field applications will be conducted in woodland tracts on residential property in southwestern (Fairfield County) and northwestern (Litchfield County) Connecticut, areas highly endemic for Lyme disease. Ticks are abunda